The accuracy of reverse genetics systems for SARS‐CoV‐2: Circular polymerase extension reaction versus bacterial artificial chromosome

Abstract Background Reverse genetics systems to rescue viruses from modified DNA are useful tools to investigate the molecular mechanisms of viruses. The COVID‐19 pandemic prompted the development of several reverse genetics systems for SARS‐CoV‐2. The circular polymerase extension reaction (CPER) method enables the rapid generation of recombinant SARS‐CoV‐2; however, such PCR‐based approaches could introduce unwanted mutations due to PCR errors. Methods To compare the accuracy of CPER and a classic reverse genetics method using bacterial artificial chromosome (BAC), SARS‐CoV‐2 Wuhan/Hu‐1/2019 was generated five times using BAC and five times using CPER. These 10 independent virus stocks were then deep sequencing, and the number of substitutions for which the frequency was greater than 10% was counted. Results No nucleotide substitutions with a frequency of greater than 10% were observed in all five independent virus stocks generated by the BAC method. In contrast, three to five unwanted nucleotide substitutions with a frequency of more than 10% were detected in four of the five virus stocks generated by the CPER. Furthermore, four substitutions with frequencies greater than 20% were generated in three virus stocks by using the CPER. Conclusions We found that the accuracy of the CPER method is lower than that of the BAC method. Our findings suggest care should be used when employing the CPER method.


| INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as a novel human pathogen in China at the end of 2019, and it continues to circulate globally. Since the World Health Organization (WHO) declared a pandemic on March 11, 2020, many variants of concern including omicron have emerged. Reverse genetics is a useful tool with which to study the functional effects of mutations. Since the emergence of SARS-CoV-2, several reverse genetics systems for SARS-CoV-2 have been reported. [1][2][3][4][5] One of them, the circular polymerase extension reaction (CPER) method, which is a PCR-based, bacterium-free method, is widely used because of its speed and simplicity. 1,2,[6][7][8][9] However, PCR-based methods come with the risk of PCR errors. In contrast, the reverse genetics system that uses bacterial artificial chromosome (BAC) is a classical method to rescue recombinant viruses possessing a large genome such as coronaviruses and herpes viruses. [10][11][12] Because BACs are amplified by Escherichia coli, the BAC method takes longer to prepare recombinant viruses than PCR-based methods. However, the BAC method using cloned and sequenceverified BACs yields recombinant viruses with a very low genetic mixture. To compare the two methods, here, we generated SARS-CoV-2 five times using the BAC method and five times using the CPER method and assessed the sequence accuracy by deep sequencing.

| BAC construction
The full-genome nucleotide sequence of SARS-CoV-2 Wuhan/Hu-1/2019 was assembled into the pBeloBAC11 vector to generate infectious cDNA clones under the control of a cytomegalovirus (CMV) promoter by using Gibson Assembly Master Mix (NEB, E2611) as described previously 14 with some modifications. Briefly, five fragments (Fa to Fe) covering the full-length SARS-CoV-2 genome with a 30-bp overlap and restriction sites and linearized pBeloBAC11 (linker fragment) were amplified by PCR using PrimeSTAR GXL DNA Polymerase (TaKaRa), which is a high fidelity PCR enzyme, 15

| CPER
Viruses were rescued by CPER as previously described. 2 In brief, the artificially synthesized DNA of SARS-CoV-2 Wuhan/Hu-1/2019 was used as a template for PCR. The six DNA fragments covering the fulllength SARS-CoV-2 genome and a linker fragment encoding hepatitis delta virus ribozyme, bovine growth hormone polyA signal, and the CMV promoter were amplified by PCR using PrimeSTAR GXL DNA Polymerase, a high fidelity PCR enzyme. The seven DNA fragments were mixed and used for CPER. 2

| Sequencing analysis
The stock viruses were subjected to next-generation sequencing using iSeq100 according to a previous report. 16 The number of substitutions whose frequency was higher than 10% was counted.

| RESULTS AND DISCUSSION
SARS-CoV-2 Wuhan/Hu-1/2019 was generated five times by using BAC and five times using CPER. In this way, we generated 10 independent virus stocks and subjected them to deep sequencing. As shown in Table 2, no nucleotide substitutions with a frequency of greater than 10% were observed in all five independent virus stocks generated by the BAC method. In contrast, 3-5 unwanted nucleotide substitutions were detected in four of the five virus stocks generated by the CPER. Although these substitutions were not dominant, four substitutions with frequencies greater than 20% were observed in three virus stocks. These results clearly demonstrate that reverse genetics by BAC is highly accurate and that the CPER method comes with a high risk of unwanted mutations.
Although the fidelity of the DNA polymerase for PCR has been improving, it is still lower than that of bacterial polymerase. Therefore, unwanted substitutions in rescued SARS-CoV-2 have been observed in some previous studies. 1,7,9 Unwanted substitutions due to PCR errors are introduced randomly and, therefore, their effects on viral characteristics cannot be estimated. This makes it difficult to explore the functions of an amino acid substitution because we want to generate a parental virus and its mutant with only that single amino acid substitution. Such an experiment requires completely identical genome sequences other than at the position of interest. Therefore, such unwanted and uncontrollable mutations might lead to incorrect conclusions that would not be drawn had the viruses been generated by using the BAC method or cloned and sequenced DNA.
The CPER method is a powerful and rapid tool to generate recombinant viruses and has been frequently used in several studies.

CONFLICT OF INTEREST STATEMENT
All the authors declared no conflicts of interest related to this work.

DATA AVAILABILITY STATEMENT
All data supporting the findings of this study are available within the paper and from the corresponding author upon request. There are no restrictions to obtaining access to the primary data.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.13109.